利用孕妇外周血中胎儿有核红细胞行无创产前性别诊断的初步研究

The Preliminary Study on Non-invasive Prenatal Gender Diagnosis Utilizing Fetal Nucleated Red Blood Cell from Maternal Blood

目的建立一种利用孕妇外周血中分选出的胎儿有核红细胞(FNRBC)进行无创伤性产前诊断的有效方法。方法收集5例妊娠7～9周的人流妇女的绒毛及外周血20ml,采用抗-CD45和抗ε链双标细胞,流式细胞仪分选CD45dim的细胞经显微挑选出ε-链免疫荧光抗体阳性的胎儿有核红细胞,再采用单细胞多重置换扩增技术(MDA)进行全基因组扩增,以扩增产物为模板,常规PCR检测Alphoid基因片段,确定胎儿性别,与绒毛PCR结果比较。结果抗-ε链、抗-CD45双标记的5例标本流式分选CD45dim细胞后,所有标本均观察到血红蛋白ε链阳性细胞。怀有2例男性和1例女性孕妇的外周血分选出的阳性细胞经MDA和PCRAlphoid基因片段扩增,显示1例全基因组扩增失败;1例诊断为男胎,1例为女性,与绒毛PCR性别检测结果完全符合。结论ε-抗体、CD45双标的CD45dim细胞经流式分选后结合显微操作技术从母血中挑选出ε-血红蛋白阳性细胞,经全基因组扩增可以有效获取足够量的研究模板,保证后续分析的准确性,可应用于性连锁遗传疾病的无创性产前诊断。

Objective The purpose of this paper is to establish an effective method using fetal nucleated red blood cells form peripheral maternal blood for non-invasive prenatal diagnosis.Methods Double-labeled mononuclear cells of 5 cases between 7 and 9 weeks＇ gestation with anti-CD45,and hemoglobin ε-chain antibody immunofluorescence before sorting by flow cytometry with CD45dim were single out anti-ε-chain positive NRBCs by micromanipulation technique.The selected cells were amplified by multiple displacement amplification（MDA）for whole genome amplification.Fetal gender was confirmed by PCR of Alphoid fragments with whole genome amplification product as a template,comparing with the sex identification by PCR of the villous.Results After sorting by flow cytometry with CD45dim,there were ε-chain of hemoglobin-positive cells in all samples.These positive cells of two male cases and one female case were selected for the whole genome amplification and subsequent detection of gender by Alphoid gene fragment amplification.The results showed that one case of whole genome amplification failed 1 case was diagnosed as male fetuses,and 1 female,which were in full compliance with the gender results of PCR with villous.Conclusion After sorting by flow cytometry with CD45dim,ε-chain of hemoglobin-positive cells were singled out for whole genome amplification,this technique can effect-ively obtain an adequate amount of template and ensure the accuracy of the follow-up analysis for sex-linked genetic diseases,non-invasive prenatal diagnosis.